3. Results and Discussion
3.2 Screening for TERRA-interacting proteins
The discovery that the human telomeric sequence is transcribed into the so-called telomeric repeat containing RNA (TERRA) by Azzalin and co-workers raised the question of the in vivo relevance of TERRA 100. TERRA consists of 5`
UUAGGG 3` tandem repeats with heterogeneous length and therefore belongs to the putative G-quadruplex forming RNA sequences 100. The possibility of this sequence to fold into a G-quadruplex structure has been shown by NMR and X-ray crystallography 206-208. Based on the finding that TERRA is co-localized at the telomeres, a regulatory and protective role of TERRA for the chromosomal ends is assumed 136. This assumption has been further strengthened by the identification of several known telomere-associated proteins found to bind to a TERRA oligonucleotide 137. Furthermore, the two members of the shelterin complex telomeric repeat factor 1 and 2 (TRF1 and TRF2) have been shown to interact with TERRA in vivo 137. However apart from this in vivo study, the situation of proteins binding to TERRA within the cell remains still unclear.
To identify proteins interacting with TERRA in vivo, a cell-based crosslinking approach was performed (Fig. 3.2.1). The idea for the use of this method evoked in relation to a publication by Hafner et al. (2010) who developed the method in order to identify RNA binding proteins and microRNA target sites and referred to as PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) 209. This method is based on the random incorporation of the uridine analog thiouridine (s4U) into cellular RNA. 4-thiouridine, in which the oxygen at position 4 of the pyrimidine ring is replaced by a sulfur atom, is photoactivatable with UV light above 320 nm wavelength 210. The UV light exposure leads to specific crosslinks between proteins, which are in close proximity to s4U containing RNA, and the RNA itself 211. Compared to conventional UV 254 nm crosslinking, the photoactivatable nucleotides should improve RNA recovery 100- to 1000-fold, using the same amount of radiation energy 209.
3. Results and Discussion
Fig. 3.2.1: Schematic representation of the cell-based crosslinking approach to purify in vivo TERRA-interacting proteins. HeLa S3 cells were fed with the photoactivatable uridine analog 4-thiouridine (s4U) (final conc. = 100 µM) for 48 h, followed by in vivo crosslinking with UV light of 365 nm (0.15 J/cm2 for 30 min). Nuclear RNA was isolated and TERRA together with crosslinked proteins were fished using magnetic beads coupled to a TERRA-specific hybridization linker DNA (NH2-(C12)-(CCCTAA)8). After the release of TERRA-crosslinked proteins by RNase treatment, the proteins were separated via SDS-gel electrophoresis. To identify the crosslinked proteins, excised protein bands were analyzed using nano-LC MS/MS.
Scheme by Dr. Kangkan Halder.
HeLa S3 cells were grown for 48 h in medium containing s4U. After random incorporation of s4U into RNA, the cells were exposed to UV light (365 nm).
Following UV crosslinking of s4U containing RNAs with interacting proteins, the nuclear extract was isolated and treated with DNase to remove DNA contaminations. To purify TERRA (TERRA-fishing), this extract was incubated with magnetic beads coupled to a TERRA-specific hybridizing linker DNA (NH2 -(C12)-(CCCTAA)8). As a control, arandomized sequence of the TERRA-specific hybridizing linker DNA was used. After washing the magnetic beads, proteins crosslinked to TERRA were released by RNase treatment. To identify the crosslinked proteins, the released proteins were separated via SDS gel electrophoresis, protein bands were excised from the SDS gel and the samples were analyzed by nano-LC-MS/MS (performed by the proteomics facility of the University of Konstanz).
Fig. 3.2.2: SDS-PAGE analysis of possible TERRA-interacting proteins. The protein eluates after TERRA-fishing were separated on a 12% SDS gel followed by silver staining. First and fourth lane: Marker (M); second lane: protein eluate after fishing with the TERRA-specific hybridization linker DNA (TERRA); third lane: protein eluate after fishing with the randomized hybridization linker DNA (Ctrl).
3. Results and Discussion
After the separation of the proteins via SDS gel electrophoresis, a silver staining of the gel was performed to visualize the protein bands (Fig. 3.2.2). At first glance differences in the pattern of the samples between the TERRA-bound proteins and the control were hardly noticeable. Nevertheless, the protein bands were excised from the gel and investigated via nano-LC MS/MS. Unfortunately, the mass spectrometry analysis of the excised protein bands revealed no usable results. According to the proteomics facility only keratin was found within the samples.
Discussion:
The analysis of the excised protein bands via nano-LC MS/MS by the proteomics facility of the University of Konstanz identified solely keratin in both eluates. This result was not expected with regard to the pattern of the silver stained SDS gel.
Although no useable results could be revealed after nano-LC MS/MS, the herein described method can be a suitable and promising approach for the detection of TERRA-interacting proteins in vivo. Therefore, it might be worth to repeat the experiment. Due to the time-consuming procedure to generate sufficient amounts of cell nuclei lysate for TERRA-fishing, it was not feasible to repeat the experiment in terms of the current study.
The situation for TERRA-interacting proteins in vivo is still quite unclear.
However based on RNA pull-down experiments followed by mass spectrometric analysis, a large number of possible TERRA-interacting proteins have been detected. As expected, most of these proteins are known telomere-associated proteins, e.g. proteins of the shelterin complex, the origin recognition complex (ORC) or proteins which belong to the heterogeneous nuclear ribonucleoprotein (HNRNP) family 137,212. More recently, Scheibe et al. (2013) performed an elegant RNA pull-down experiment combined with stable isotope labeling by amino acids in cell culture (SILAC), thereby allowing also protein quantification
213. In addition to the proteins found in previous studies, they were able to identify 15 novel TERRA-interacting proteins. Based on endoribonuclease-prepared short-interference RNA (esiRNA) knock-down experiments they found that SRRT/ARS2, which is known to be involved in miRNA processing, and
MORF4L2, a component of the NuA4 histone acetyltransferase complex, are involved in the regulation of detectable total TERRA as well as telomere-bound TERRA amounts. Although the herein mentioned RNA pull-down techniques seem well-suited for the identification of TERRA-interacting proteins, they all require nuclei lysis prior to the “fishing” of TERRA-interacting proteins. This lysis step might influence possible TERRA-protein interactions and therefore differ to the situation in vivo. It is therefore conceivable to lose possible protein-RNA interactions. Furthermore, these RNA pull-down techniques might be limited by the requirement of kinetically stable interactions between the respective RNA and the bound proteins and therefore may also lead to a loss of less stable protein-RNA interactions. To that end, the s4U-based screening approach might be helpful to overcome this issue because the crosslinking of proteins to RNA occurs prior to the nuclei lysis and stable RNA-protein interactions are not required.
3. Results and Discussion